Standard selection treatments with sulfadiazine limit Plasmodium yoelii host-to-vector transmission

Some early antimalarial drugs have been repurposed for experimental applications, thus extending their utility well beyond the point when resistance becomes prevalent in circulating parasite populations. One such drug is sulfadiazine, which is an analog of p-aminobenzoic acid (pABA), and acts as a competitive inhibitor of dihydropteroate synthase, which is an essential enzyme in the parasite's folate synthesis pathway that is required for DNA synthesis. Sulfadiazine treatment of mice infected with P. yoelii and P. berghei is routinely used to enrich for gametocytes by killing asexual blood stage parasites, but it is not well known if the exposed gametocytes are perturbed or if there is a detrimental effect on transmission. To determine if there was a significant effect of sulfadiazine exposure upon host-to-vector transmission, we transmitted Plasmodium yoelii (17XNL strain) parasites to Anopheles stephensi mosquitoes and evaluated the prevalence of infection (percent of mosquitoes infected) and intensity of infection (number of oocysts per infected mosquito) under different sulfadiazine treatment conditions of the mouse or of the mosquitoes. We observed that parasites exposed to sulfadiazine either in the mouse host or in the mosquito vector had a reduction in both the number of mosquitoes that became infected and in the intensity of infection compared to untreated controls. We also observed that provision of freshly prepared pABA in the mosquito sugar water could only marginally overcome the defects caused by sulfadiazine treatment. In contrast, we determined that gametocytes exposed to sulfadiazine were able to be fertilized and develop into morphologically mature ookinetes in vitro, and thus that sulfadiazine exposure in the host may be reversible if the drug is washed out and the parasites are supplemented with pABA in the culture media. Overall, this indicates that sulfadiazine dampens host-to-vector transmission, and that this inhibition can only be partially overcome by exposure to fresh pABA in vivo and in vitro. Because gametocytes are of great interest for developing transmission blocking interventions, we recommend that less disruptive approaches for gametocyte enrichment be used in order to study minimally perturbed parasites.

The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts

Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites is still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to safely enter the mosquito salivary glands without causing cell damage, to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.

Assessment in vitro of the antimalarial and transmission blocking activities of Cipargamin and Ganaplacide in artemisinin resistant Plasmodium falciparum

Artemisinin resistance in Plasmodium falciparum has emerged and spread widely in the Greater Mekong Subregion threatening current first line artemisinin combination treatments. New antimalarial drugs are needed urgently. Cipargamin (KAE609) and ganaplacide (KAF156) are promising novel antimalarial compounds in advanced stages of development. Both compounds have potent asexual blood stage activities, inhibit P. falciparum gametocytogenesis and reduce oocyst development in anopheline mosquitoes. In this study, we compared the asexual and sexual stage activities of cipargamin, ganaplacide and artesunate in artemisinin resistant P. falciparum isolates (N=7, K13 mutation; C580Y, G449A and R539T) from Thailand and Cambodia. Asexual blood stage antimalarial activity was evaluated in a SYBR-green I based 72h in vitro assay, and the effects on male and female mature stage V gametocytes were assessed in the P. falciparum dual gamete formation assay. Ganaplacide had higher activities when compared to cipargamin and artesunate, with a mean (SD) IC50 against asexual stages of 5.5 (1.1) nM, 7.8 (3.9) nM for male gametocytes and 57.9 (59.6) nM for female gametocytes. Cipargamin had a similar potency against male and female gametocytes, with a mean (SD) IC50 of 123.1 (80.2) nM for male gametocytes, 88.5 (52.7) nM for female gametocytes and 2.4 (0.6) nM for asexual stages. Both cipargamin and ganaplacide showed significant transmission-blocking activities against artemisinin resistant P. falciparum in vitro .

New insights into antimalarial chemopreventive activity of antifolates

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, the thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, have yet to be achieved. Using a transgenic P. berghei harboring quadruple mutant dhfr from P. falciparum (Pb::Pfdhfr -4M ) , we demonstrate that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not to a new class of antifolate designed to overcome the resistance such as P218. Detailed investigation to pin-point stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data shows that P218 also inhibited the late hepatic stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg, P218 prevented infection from up to 25,000 pyrimethamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates, as a chemopreventive therapeutic which could lead to a more efficient and cost effective treatment regime.

Deletion of Plasmodium falciparum ubc13 increases parasite sensitivity to the mutagen, methyl methanesulfonate and dihydroartemisinin

The inducible Di-Cre system was used to delete the putative ubiquitin-conjugating enzyme 13 gene (ubc13) of Plasmodium falciparum to study its role in ubiquitylation and the functional consequence during the parasite asexual blood stage. Deletion resulted in a significant reduction of parasite growth in vitro, reduced ubiquitylation of the Lys63 residue of ubiquitin attached to protein substrates, and an increased sensitivity of the parasite to both the mutagen, methyl methanesulfonate and the antimalarial drug dihydroartemisinin (DHA), but not chloroquine. The parasite was also sensitive to the UBC13 inhibitor NSC697923. The data suggest that this gene does code for an ubiquitin conjugating enzyme responsible for K63 ubiquitylation, which is important in DNA repair pathways as was previously demonstrated in other organisms. The increased parasite sensitivity to DHA in the absence of ubc13 function indicates that DHA may act primarily through this pathway and that inhibitors of UBC13 may both enhance the efficacy of this antimalarial drug and directly inhibit parasite growth.

Studies of Potency and Efficacy of an Optimized Artemisinin-Quinoline Hybrid against Multiple Stages of the Plasmodium Life Cycle

A recently developed artemisinin-quinoline hybrid, named 163A, has been shown to display potent activity against the asexual blood stage of Plasmodium, the malaria parasite. In this study, we determined its in vitro cytotoxicity to mammalian cells, its potency to suppress P. berghei hepatic infection and to decrease the viability of P. falciparum gametocytes, in addition to determining whether the drug exhibits efficacy of a P. berghei infection in mice. This hybrid compound has a low level of cytotoxicity to mammalian cells and, conversely, a high level of selectivity. It is potent in the prevention of hepatic stage development as well as in killing gametocytes, denoting a potential blockage of malaria transmission. The hybrid presents a potent inhibitory activity for beta-hematin crystal formation, in which subsequent assays revealed that its endoperoxide component undergoes bioactivation by reductive reaction with ferrous heme towards the formation of heme-drug adducts; in parallel, the 7-chloroquinoline component has binding affinity for ferric hemin. Both structural components of the hybrid co-operate to enhance the inhibition of beta-hematin, and this bitopic ligand property is essential for arresting the growth of asexual blood parasites. We demonstrated the in vivo efficacy of the hybrid as an erythrocytic schizonticide agent in comparison to a chloroquine/artemisinin combination therapy. Collectively, the findings suggest that the bitopic property of the hybrid is highly operative on heme detoxification suppression, and this provides compelling evidence for explaining the action of the hybrid on the asexual blood stage. For sporozoite and gametocyte stages, the hybrid conserves the potency typically observed for endoperoxide drugs, and this is possibly achieved due to the redox chemistry of endoperoxide components with ferrous heme.

Plasmodium falciparum infected humanized mice: a viable preclinical tool

Summary Extensive research conducted on mouse–human chimeras has advanced our understanding on infectious diseases including the human–malaria parasite, Plasmodium falciparum ( P. falciparum). In vitro culture of asexual-blood stage infection of P. falciparum does not answer all questions related to parasitology, pharmacology and immunology, and complex life cycle, complicated genome, evolution of drug resistance and poor diagnosis makes it difficult to understand the patho-biology of parasite. Unavailability of effective-vaccine and issues of drug resistance advocates the use of human cell/tissues reconstituted immunodeficient-mice to P. falciparum. A number of immunodeficient-strains (TK/NOG, FRG/NOD, NOD/SCID/IL-2 receptor γ chain null, NOD severe combined immunodeficiency gamma [NSG] mouse, NOD.Rag1−/− IL2Rγ−/− [NRG; DRAG]) are used for humanization purposes. Additionally, human-hematopoietic stem cells (CD34 reconstituted-NSG [human immune system]) mice support the engraftment and repopulation of immune effecters to study systemic inflammatory diseases.

Systemic host inflammation induces stage-specific transcriptomic modification and slower maturation in malaria parasites

Maturation rate of malaria parasites within red blood cells (RBC) can be influenced by host nutrient status or circadian rhythm. Here, we observed in mice that systemic host inflammation, induced by lipopolysaccharide (LPS) conditioning or ongoing acute malaria infection, slowed the progression of a single cohort of parasites from one generation of RBC to the next. LPS-conditioning and acute infection both triggered substantial changes to the metabolomic composition of plasma in which parasites circulated. This altered plasma directly slowed parasite maturation in a manner that could not be rescued by supplementation, consistent with the presence of inhibitory factors. Single-cell transcriptomic assessment of mixed parasite populations, exposed to a short period of systemic host inflammation in vivo, revealed specific impairment in the transcriptional activity and translational capacity of trophozoites compared to rings or schizonts. Thus, we provide in vivo evidence of transcriptomic and phenotypic plasticity of asexual blood-stage Plasmodium parasites when exposed to systemic host inflammation.

New insights into antimalarial chemopreventive activity of antifolates

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, the thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, have yet to be achieved. Using a transgenic P. berghei harboring quadruple mutant dhfr from P. falciparum (Pb::Pfdhfr-4M), we demonstrate that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not a new class of antifolate designed to overcome the resistance such as P218. Detailed investigation to pin-point stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data shows that P218 also inhibited the late hepatic stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg, P218 prevented infection from up to 25,000 pyrimentamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates, as a chemopreventive therapeutic which could lead to a more efficient and cost effective treatment regime.

Exploratory analysis of the effect of helminth infection on the immunogenicity and efficacy of the asexual blood-stage malaria vaccine candidate GMZ2

Background Helminths can modulate the host immune response to Plasmodium falciparum and can therefore affect the risk of clinical malaria. We assessed here the effect of helminth infections on both the immunogenicity and efficacy of the GMZ2 malaria vaccine candidate, a recombinant protein consisting of conserved domains of GLURP and MSP3, two asexual blood-stage antigens of P. falciparum. Controlled human malaria infection (CHMI) was used to assess the efficacy of the vaccine. Methodology In a randomized, double-blind Phase I clinical trial, fifty, healthy, lifelong malaria-exposed adult volunteers received three doses of GMZ2 adjuvanted with either Cationic Adjuvant Formulation (CAF) 01 or Alhydrogel, or a control vaccine (Rabies) on days (D) 0, D28 and D56, followed by direct venous inoculation (DVI) of 3,200 P. falciparum sporozoites (PfSPZ Challenge) approximately 13 weeks after last vaccination to assess vaccine efficacy. Participants were followed-up on a daily basis with clinical examinations and thick blood smears to monitor P. falciparum parasitemia for 35 days. Malaria was defined as the presence of P. falciparum parasites in the blood associated with at least one symptom that can be associated to malaria over 35 days following DVI of PfSPZ Challenge. Soil-transmitted helminth (STH) infection was assessed by microscopy and by polymerase chain reaction (PCR) on stool, and Schistosoma infection was assessed by microscopy on urine. Participants were considered as infected if positive for any helminth either by PCR and/or microscopy at D0 and/or at D84 (Helm+) and were classified as mono-infection or co-infection. Total vaccine-specific IgG concentrations assessed on D84 were analysed as immunogenicity outcome. Main findings The helminth in mono-infection, particularly Schistosoma haematobium and STH were significantly associated with earlier malaria episodes following CHMI, while no association was found in case of coinfection. In further analyses, the anti-GMZ2 IgG concentration on D84 was significantly higher in the S. haematobium-infected and significantly lower in the Strongyloides stercoralis-infected groups, compared to helminth-negative volunteers. Interesting, in the absence of helminth infection, a high anti-GMZ2 IgG concentration on D84 was significantly associated with protection against malaria. Conclusions Our results suggest that helminth infection may reduce naturally acquired and vaccine-induced protection against malaria. Vaccine-specific antibody concentrations on D84 may be associated with protection in participants with no helminth infection. These results suggest that helminth infection affect malaria vaccine immunogenicity and efficacy in helminth endemic countries.